RNA molecules with structure-dependent functions play important roles throughout molecular biology. The broad, long-term objective of the proposed research is to better understand the structural and catalytic properties of RNA. The focus is on two ribozymes at the frontier of RNA catalytic function. One ribozyme ligates RNA and is among the fastest known RNA catalysts. The other ribozyme polymerizes RNA using the same reaction as that of protein enzymes that replicate RNA. This reaction is among the most complex reactions known to be catalyzed by RNA. The reactions and structures of these two ribozymes will be examined and enhanced with a set of molecular biology experiments that involve biochemical, engineering, and combinatorial approaches. Specific aims are 1) to characterize and optimize the rapid folding and catalysis of the RNA ligase ribozyme, 2) to examine the tertiary structure of the ligase ribozyme, and 3) to optimize and characterize the polymerase ribozyme. These experiments will contribute to our fundamental knowledge of RNA molecular biology. They will describe and extend known limits of RNA catalytic function, lay the foundation for later studies exploring how RNA can achieve such rapid and complex function, and speak to the ability of RNA to catalyze self- replication-the central presumption of current theories of the early evolution of life. Innovations and insights from these experiments will also enable and inspire those seeking to isolate new ribozymes with efficient and complex functions, including ribozymes useful as diagnostics, therapeutics, and research tools.